M Richardson

Fundação Ezequiel Dias, Camelleira, Pernambuco, Brazil

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Publications (23)59.75 Total impact

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    ABSTRACT: PnTx3-4 is a toxin isolated from the venom of the spider Phoneutria nigriventer that blocks N-, P/Q-, and R-type voltage-gated calcium channels and has great potential for clinical applications. In this report we used the SUMO system to express large amounts of recombinant PnTx3-4 peptide, which was found in both soluble and insoluble fractions of bacterial extracts. We purified the recombinant toxin from both fractions and showed that the recombinant peptide showed biological activity similar to the native PnTx3-4. In silico analysis of the primary sequence of PnTx3-4 indicated that the peptide conforms to all the criteria of a knottin scaffold. Additionally, circular dichroism spectrum analysis of the recombinant PnTx3-4 predicted that the toxin structure is composed of approximately 53% turns/unordered, 31% α-helix and 16% β-strand, which is consistent with predicted model of the PnTx3-4 knottin scaffold available at the knottin database (http://knottin.cbs.cnrs.fr). These studies provide the basis for future large scale production and structure-function investigation of PnTx3-4.
    Toxicon 05/2012; 60(5):907-18. · 2.92 Impact Factor
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    ABSTRACT: Erectile dysfunction (ED) mechanisms in diabetic patients are multifactorial and often lead to resistance to current therapy. Animal toxins have been used as pharmacological tools to study penile erection. Human accidents involving the venom of Phoneutria nigriventer spider are characterized by priapism. We hypothesize that PnTx2-6 potentiates cavernosal relaxation in diabetic mice by increasing cyclic guanosine monophosphate (cGMP). This effect is neuronal nitric oxide synthase (nNOS) dependent. Cavernosal strips were contracted with phenylephrine (10(-5) M) and relaxed by electrical field stimulation (20 V, 1-32 Hz) in the presence or absence of PnTx2-6 (10(-8) M). Cavernosal strips from nNOS- and endothelial nitric oxide synthase (eNOS)-knockout (KO) mice, besides nNOS inhibitor (10(-5) M), were used to evaluate the role of this enzyme in the potentiation effect evoked by PnTx2-6. Tissue cGMP levels were determined after stimulation with PnTx2-6 in presence or absence of N-nitro-L-arginine methyl ester (L-NAME) (10(-4) M) and ω-conotoxin GVIA (10(-6) M), an N-type calcium channel inhibitor. Results showed that PnTx2-6 enhanced cavernosal relaxation in diabetic mice (65%) and eNOS KO mice, but not in nNOS KO mice. The toxin effect in the cavernosal relaxation was abolished by nNOS inhibitor. cGMP levels are increased by PnTx2-6, however, L-NAME abolished this enhancement as well as ω-conotoxin GVIA. We conclude that PnTx2-6 facilitates penile relaxation in diabetic mice through a mechanism dependent on nNOS, probably via increasing nitric oxide/cGMP production.
    International journal of impotence research 10/2011; 24(2):69-76. · 2.73 Impact Factor
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    ABSTRACT: Erectile dysfunction (ED) mechanisms in diabetic patients are multifactorial and often lead to resistance to current therapy. Animal toxins have been used as pharmacological tools to study penile erection. Human accidents involving the venom of Phoneutria nigriventer spider are characterized by priapism. We hypothesize that PnTx2-6 potentiates cavernosal relaxation in diabetic mice by increasing cyclic guanosine monophosphate (cGMP). This effect is neuronal nitric oxide synthase (nNOS) dependent. Cavernosal strips were contracted with phenylephrine (10(-5) M) and relaxed by electrical field stimulation (20 V, 1-32 Hz) in the presence or absence of PnTx2-6 (10(-8) M). Cavernosal strips from nNOS- and endothelial nitric oxide synthase (eNOS)-knockout (KO) mice, besides nNOS inhibitor (10(-5) M), were used to evaluate the role of this enzyme in the potentiation effect evoked by PnTx2-6. Tissue cGMP levels were determined after stimulation with PnTx2-6 in presence or absence of N-nitro-L-arginine methyl ester (L-NAME) (10(-4) M) and ω-conotoxin GVIA (10(-6) M), an N-type calcium channel inhibitor. Results showed that PnTx2-6 enhanced cavernosal relaxation in diabetic mice (65%) and eNOS KO mice, but not in nNOS KO mice. The toxin effect in the cavernosal relaxation was abolished by nNOS inhibitor. cGMP levels are increased by PnTx2-6, however, L-NAME abolished this enhancement as well as ω-conotoxin GVIA. We conclude that PnTx2-6 facilitates penile relaxation in diabetic mice through a mechanism dependent on nNOS, probably via increasing nitric oxide/cGMP production.
    International Journal of Impotence Research 10/2011; 24(2):69-76. · 1.51 Impact Factor
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    ABSTRACT: Phα1β is a potent toxin obtained from the spider Phoneutria nigriventer that blocks neuronal voltage-sensitive Ca2+ channels. This study compared the antiallodynic effects of Phα1β, ω-conotoxin MVIIA and morphine in mice and their side effects in rats. Mechanical allodynia was measured in mice receiving single intrathecal administration of Phα1β, ω-conotoxin MVIIA or morphine before or after the incisional plantar procedure. The effect of the treatments on cardiovascular profile and global neurological were evaluated in rats. The expression of pro or anti-inflammatory cytokines of human polymorph mononuclear cells was also evaluated. Preemptive use of ω-conotoxin MVIIA (1.0 or 10 pmol/site) or morphine (1000 pmol/site) induced shorter antiallodynic effect than Phα1β (100 pmol/site) in mice. Post-incision administration of Phα1β (200 pmol/site) induced longer mechanical antiallodynic effect than ω-conotoxin MVIIA (1.0 or 10 pmol/site) or morphine (1000 pmol/site). Intrathecal injection of Phα1β (200 pmol/site) and morphine (433 pmol/site) did not change while ω-conotoxin MVIIA (100 pmol/site) increased the heart rate in rats 3 h after its administration. Phα1β (200 pmol/site), ω-conotoxin MVIIA (100 pmol/site) and morphine (433 pmol/site) did not change mean arterial pressure 0.5 and 3 h after their administration. The treatments did not alter neurological performance assessed by global neurological evaluation and open-field test. The tested drugs did not induced expression of pro or anti-inflammatory cytokines in CD4 monocytes. In conclusion, preemptive administration Phα1β in mice induced longer antiallodynic effect than ω-conotoxin MVIIA and morphine. Phα1β also induced a longer mechanical antiallodynic effect than ω-conotoxin MVIIA and morphine when used after the surgical incision. The present results suggest that Phα1β has a potential application in the management of postoperative pain with low side effects.Highlights► Phα1β induced longer postoperative pain reduction effect than ω-conotoxin MVIIA or morphine. ► The drugs did not change mean arterial pressure after intrathechal injection. ► Intrathecal administration of ω-conotoxin MVIIA increased the heart rate while Phα1β or morphine had no effect. ► The drugs did not alter neurological performance and the expression of pro or anti-inflammatory cytokines. ► Phα1β has a potential application in the management of postoperative pain with low side effects.
    Toxicon 09/2011; 58(8):626-633. · 2.92 Impact Factor
  • European Journal of Pain Supplements 01/2011; 5(1):26-26.
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    ABSTRACT: Studies revealed that the venom of the Brazilian "armed" spider Phoneutria nigriventer contains potent neurotoxins that caused excitatory symptoms such as salivation, lachrymation, priapism, convulsions, flaccid and spastic paralysis. It was also reported that the main mechanism of action of those neurotoxins are effects on ion channels such as inhibition of the inactivation of Na+ channels, blockage of K+ channels and blockage of calcium channels. The venom from Phoneutria keyserlingi, as might be expected, contains a series of polypeptides that are very similar, but not identical, to the proteins previously obtained from the venom of P. nigriventer in terms of their amino acid sequences and biological activities. We evaluated the effects of some of the toxins of P. nigriventer and P. keyserlingi on glutamate release and the decrease in [Ca2+]i by using synaptosomes of rat brain cortices and fluorimetric assays. Sequence comparisons between the Phoneutria toxins of both the species showed great similarity in the location of cysteine residues. However, thus far, no pharmacological assays were performed to evaluate the extension of those biochemical modifications. Our results showed that differences between the amino acid sequences of Phoneutria toxins of both the species lead to the significant changes in the pharmacological properties of these toxins.
    Cellular and molecular biology (Noisy-le-Grand, France) 01/2010; 56 Suppl:OL1223-30. · 1.46 Impact Factor
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    ABSTRACT: The venom of the spider Phoneutria nigriventer contains several toxins that have bioactivity in mammals and insects. Accidents involving humans are characterized by various symptoms including penile erection. Here we investigated the action of Tx2-6, a toxin purified from the P. nigriventer spider venom that causes priapism in rats and mice. Erectile function was evaluated through changes in intracavernosal pressure/mean arterial pressure ratio (ICP/MAP) during electrical stimulation of the major pelvic ganglion (MPG) of normotensive and deoxycorticosterone-acetate (DOCA)-salt hypertensive rats. Nitric oxide (NO) release was detected in cavernosum slices with fluorescent dye (DAF-FM) and confocal microscopy. The effect of Tx2-6 was also characterized after intracavernosal injection of a non-selective nitric oxide synthase (NOS) inhibitor, L-NAME. Subcutaneous or intravenous injection of Tx2-6 potentiated the elevation of ICP/MAP induced by ganglionic stimulation. L-NAME inhibited penile erection and treatment with Tx2-6 was unable to reverse this inhibition. Tx2-6 treatment induced a significant increase of NO release in cavernosum tissue. Attenuated erectile function of DOCA-salt hypertensive rats was fully restored after toxin injection. Tx2-6 enhanced erectile function in normotensive and DOCA-salt hypertensive rats, via the NO pathway. Our studies suggest that Tx2-6 could be important for development of new pharmacological agents for treatment of erectile dysfunction.
    Toxicon 07/2008; 51(7):1197-206. · 2.92 Impact Factor
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    ABSTRACT: The venom of the spider Phoneutria nigriventer contains several toxins that have bioactivity in mammals and insects. Accidents involving humans are characterized by various symptoms including penile erection. Here we investigated the action of Tx2-6, a toxin purified from the P. nigriventer spider venom that causes priapism in rats and mice. Erectile function was evaluated through changes in intracavernosal pressure/mean arterial pressure ratio (ICP/MAP) during electrical stimulation of the major pelvic ganglion (MPG) of normotensive and deoxycorticosterone-acetate (DOCA)-salt hypertensive rats. Nitric oxide (NO) release was detected in cavernosum slices with fluorescent dye (DAF-FM) and confocal microscopy. The effect of Tx2-6 was also characterized after intracavernosal injection of a non-selective nitric oxide synthase (NOS) inhibitor, L-NAME. Subcutaneous or intravenous injection of Tx2-6 potentiated the elevation of ICP/MAP induced by ganglionic stimulation. L-NAME inhibited penile erection and treatment with Tx2-6 was unable to reverse this inhibition. Tx2-6 treatment induced a significant increase of NO release in cavernosum tissue. Attenuated erectile function of DOCA-salt hypertensive rats was fully restored after toxin injection. Tx2-6 enhanced erectile function in normotensive and DOCA-salt hypertensive rats, via the NO pathway. Our studies suggest that Tx2-6 could be important for development of new pharmacological agents for treatment of erectile dysfunction.
    Toxicon 02/2008; 51(7):1197-2006. · 2.92 Impact Factor
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    ABSTRACT: A family of 4kDa neurotoxic peptides was purified from venoms of Phoneutria spiders. All have six cysteine residues, and low similarity with other neurotoxins. Three toxins caused moderate inhibition of L-type Ca(2+) channels. The structure of toxin PRTx27C3 was modeled and compared with toxin ADO1. The importance of four residues is suggested.
    Protein and Peptide Letters 02/2008; 15(7):700-8. · 1.99 Impact Factor
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    ABSTRACT: Various neurotoxins have been described from the venom of the Brazilian spider Phoneutria nigriventer, but little is known about the venoms of the other species of this genus. In the present work, we describe the purification and some structural and pharmacological features of a new toxin (PRTx3-7) from Phoneutria reidyi that causes flaccid paralysis in mice. The observed molecular mass (4627.26 Da) was in accordance with the calculated mass for the amidated form of the amino acid sequence (4627.08 Da). The presence of an alpha-amidated C-terminus was confirmed by MS/MS analysis of the C-terminal peptide, isolated after enzymatic digestion of the native protein with Glu-C endoproteinase. The purified protein was injected (intracerebro-ventricular) into mice at dose levels of 5 microg/mouse causing immediate agitation and clockwise gyration, followed by the gradual development of general flaccid paralysis. PRTx3-7 at 1 microM inhibited by 20% the KCl-induced increase on [Ca2+]i in rat brain synaptosomes. The HEK cells permanently expressing L, N, P/Q and R HVA Ca2+ channels were also used to better characterize the pharmacological features of PRTx3-7. To our surprise, PRTx3-7 shifted the voltage-dependence for activation towards hyperpolarized membrane potentials for L (-4 mV), P/Q (-8 mV) and R (-5 mV) type Ca2+ currents. In addition, the new toxin also affected the steady state of inactivation of L-, N- and P/Q-type Ca2+ currents.
    Cellular and Molecular Neurobiology 03/2007; 27(1):129-46. · 2.29 Impact Factor
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    ABSTRACT: Arachnids have a venom apparatus and secrete a complex chemical mixture of low molecular mass organic molecules, enzymes and polypeptide neurotoxins designed to paralyze or kill their prey. Most of these toxins are specific for membrane voltage-gated sodium channels, although some may also target calcium or potassium channels and other membrane receptors. Scorpions and spiders have provided the greatest number of the neurotoxins studied so far, for which, a good number of primary and 3D structures have been obtained. Structural features, comprising a folding that determines a similar spatial distribution of charged and hydrophobic side chains of specific amino acids, are strikingly common among the toxins from spider and scorpion venoms. Such similarities are, in turn, the key feature to target and bind these proteins to ionic channels. The search for new insecticidal compounds, as well as the study of their modes of action, constitutes a current approach to rationally design novel insecticides. This goal tends to be more relevant if the resistance to the conventional chemical products is considered. A promising alternative seems to be the biotechnological approach using toxin-expressing recombinant baculovirus. Spider and scorpion toxins having insecticidal activity are reviewed here considering their structures, toxicities and action mechanisms in sodium channels of excitable membranes.
    Comparative Biochemistry and Physiology Part C Toxicology & Pharmacology 10/2006; 146(1-2):264-79. · 2.71 Impact Factor
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    ABSTRACT: A new insecticidal toxin Tx4(5-5) was isolated from the fraction PhTx4 of the venom of the spider Phoneutria nigriventer by reverse phase high performance liquid chromatography (HPLC) and anion exchange HPLC. The complete amino acid sequence determined by automated Edman degradation showed that Tx4(5-5) is a single chain polypeptide composed of 47 amino acid residues, including 10 cysteines, with a calculated molecular mass of 5175 Da. Tx4(5-5) shows 64% of sequence identity with Tx4(6-1), another insecticidal toxin from the same venom. Tx4(5-5) was highly toxic to house fly (Musca domestica), cockroach (Periplaneta americana) and cricket (Acheta domesticus ), producing neurotoxic effects (knock-down, trembling with uncoordinated movements) at doses as low as 50 ng/g (house fly), 250 ng/g (cockroach) and 150 ng/g (cricket). In contrast, intracerebroventricular injections (30 microg) into mice induced no behavioural effects. Preliminary electrophysiological studies carried out on whole-cell voltage-clamped rat hippocampal neurones indicated that Tx4(5-5) (at 1 microM) reversibly inhibited the N-methyl-D-aspartate-subtype of ionotropic glutamate receptor, while having little or no effect on kainate-, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid- or gamma-aminobutyric acid-activated currents.
    Toxicon 01/2001; 39(2-3):309-17. · 2.92 Impact Factor
  • Toxicon 01/1996; 34(1):19-20. · 2.92 Impact Factor
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    ABSTRACT: An insecticidal peptide referred to as Tx4(6-1) was purified from the venom of the spider Phoneutria nigriventer by a combination of gel filtration, reverse-phase fast liquid chromatography on Pep-RPC, reverse-phase high performance liquid chromatography (HPLC) on Vydac C18 and ion-exchange HPLC on cationic columns. Tx4(6-1) is highly toxic to house flies. At levels of 0.5 ng/house fly it caused excitatory symptoms immediately after intrathoracical injection. However, in mice injections of 0.03 mg of the toxin intracerebroventricularly resulted in no apparent symptoms of intoxication. These results demonstrate that Tx4(6-1) of P. nigriventer has no toxicity for mice, and suggest that it is a specific anti-insect toxin. The mol. wt (5244.6) and purity of the toxin were determined by desorption mass spectroscopy. The complete amino acid sequence of this toxin was established by direct automated Edman degradation and manual 4-N,N'-dimethylaminoazobenzene-4'isothiocyanate/phenyl-isothiocyanate microsequence analyses of peptides obtained from digests with various proteases. The protein contains 48 amino acids including 10 Cys and 6 Lys. The N-terminal and C-terminal residues were Cys. The Tx4(6-1) sequence differs from that of previously characterized neurotoxins found in the same and other venom spiders, but exhibited sequence similarities in the location of the Cys residues.
    Toxicon 02/1995; 33(1):83-93. · 2.92 Impact Factor
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    ABSTRACT: The complete amino acid sequence of a thrombin-like enzyme with gyroxin activity isolated from the venom of the bushmaster snake Lachesis muta muta was determined by automated and DABITC/PITC microsequencing of the intact protein; fragments derived from it by separate cleavages with cyanogen bromide, iodosobenzoic acid and hydroxylamine; and peptides resulting from enzymatic digestions with trypsin, pepsin, chymotrypsin, and elastase. The protein, which is composed of 228 residues, contains four putative sites of N-linked glycosylation and exhibits significant sequence similarities with other serine proteases reported from snake venoms.
    FEBS Letters 09/1993; 329(1-2):116-20. · 3.58 Impact Factor
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    ABSTRACT: Six neurotoxic peptides (Tx3-1 to Tx3-6) were purified from the venom of the spider Phoneutria nigriventer by a combination of gel filtration, reverse phase FPLC on PEP-RPC and PRO-RPC columns, reverse phase HPLC on Vydac C18, and ion exchange HPLC on cationic and anionic columns. These toxins caused different neurological symptoms in mice after intracerebroventricular injection. At dose levels of 5 micrograms/mouse, Tx3-3 and Tx3-4 caused rapid general flaccid paralysis followed by death in 10-30 min; Tx3-2 induced immediate clockwise gyration and flaccid paralysis after 6 hr; Tx3-1, Tx3-5 and Tx3-6 produced paralysis only in the posterior limbs and gradual decreases in movement and aggression during 24 hr. The mol. wt of these cystine-rich peptides were found to be in the range of 3500-8500 by mass spectroscopy and SDS-PAGE. The complete amino acid sequences of the neurotoxins Tx3-1 (40 residues), Tx3-2 (34 residues) and Tx3-6 (55 residues), and the N-terminal sequences of Tx3-3 (34 res.), Tx3-4 (40 res.) and Tx3-5 (36 res.) were established by direct automated Edman degradation, and manual DABITC/PITC microsequence analyses of peptides obtained from digests with various proteases. The structures of these Tx3 neurotoxins from Phoneutria nigriventer exhibited sequence similarities to one another and to the neurotoxins from the venoms of the spiders Hololena curta and Agelenopsis aperta, which were most evident in the location of the Cys residues.
    Toxicon 02/1993; 31(1):35-42. · 2.92 Impact Factor
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    ABSTRACT: Four neurotoxic polypeptides (Tx2-1, Txt2-5, Tx2-6 and Tx2-9) were purified from the venom of the South American 'armed' spider Phoneutria nigriventer (Keys) by gel filtration and reverse phase FPLC and HPLC. These cysteine-rich polypeptides exhibited different levels of neurotoxicity in mice after intracerebroventricular injection. Tx2-1, Tx2-5 and Tx2-6 caused spastic paralysis and death, but the less toxic Tx2-9 produced only tail erection and scratching. The molecular weights of the polypeptides as determined by desorption mass spectroscoopy were 5838.8 for Tx2-1, 5116.6 (Tx2-5), 5291.3 (Tx2-6) and 3742.1 (Tx2-9). The complete amino acid sequences of the neurotoxins were determined by automated Edman degradation and by manual DABITC-PITC microsequence analysis of peptides obtained after digestions with various proteases. The amino acid sequences of Tx2-1 (53 residues), Tx2-5 (49 residues) and Tx2-6 (48 residues) were homologous, but had only limited similarities to the less toxic Tx2-9 (32 residues). All four polypeptides had varying sequence identities with other neurotoxins from different spider species and biologically active peptides from scorpions, a sea snail and seeds of Mirabilis jalapa.
    FEBS Letters 10/1992; 310(2):153-6. · 3.58 Impact Factor
  • K P Häger, U Jensen, J Gilroy, M Richardson
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    ABSTRACT: The sequence of the first 52 amino acids at the N-terminus of the beta-subunit of a legumin-like protein from seeds of the gymnosperm Ginkgo biloba were determined by automated sequencing and DABITC/PITC microsequence analyses of peptides derived from the protein by enzymatic digestions and chemical cleavage with CNBr. The protein from Ginkgo exhibits sequence homologies (32-49% identities) with the 11S globulins and legume-like proteins from seeds of various angiosperm monocotyledons and dicotyledons.
    Phytochemistry 03/1992; 31(2):523-5. · 3.05 Impact Factor
  • C Bloch, M Richardson
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    ABSTRACT: Two inhibitors (SI alpha 4 and SI alpha 5) of the alpha-amylases from insect and mammalian sources were purified from seeds of Sorghum bicolor by saline extraction, precipitation with ammonium sulphate, affinity chromatography on Red Sepharose, and preparative and analytical reverse-phase HPLC on columns of Vydac C18. The complete primary structures of these two inhibitors were determined by automated degradation of the intact, reduced and S-alkylated proteins and by manual 4-N,N-dimethylaminoazobenzene-4-isothiocyanate/phenyl isothiocyanate microsequencing of peptides derived from them following enzyme digests. The amino acid sequences were as follows: SI alpha 4: TVDVTACAPGLAIPAPPLPTCRTFARPRTCGLGGPYGPVDPSPVLKQ- RCCRELAAVPSRCRCAALGFMMDGVDAPLQDFRGCTREMQRIYAVSRLTRAAECNLPTIPGGGCHLSNS PR; and SI alpha 5: ANWCEPGLVIPLNPLPSCRTYMVRRACGVSIGPVVPLPVLKERCCSELEKLV- PYCRCGALRTALDSMMTGYEMRPTCSWGGLLTFAPTIVCYRECNLRTLHGRPFCYALGAEGTTT. Comparisons of these sequences with one another and with those of other proteins in the US National Biomedical Research Foundation Databank indicated that the two Sorghum proteins had significant similarities (21%-42% identity) with the members of the cereal superfamily of enzyme inhibitors.
    Protein sequences & data analysis 02/1992; 5(1):27-30.
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    E F Sanchez, C R Diniz, M Richardson
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    ABSTRACT: The complete amino acid sequence the haemorrhagic agent LHFII, a Zn and Ca containing metalloproteinase isolated from the venom of the Bushmaster snake Lachesis muta muta was determined by automated and DABITC/PITC microsequencing of the intact protein, fragments derived by cleavage with cyanogen bromide, and peptides resulting from enzymatic digestions with trypsin and the protease from S. aureus V8. The protein is composed of 200 residues and exhibits considerable sequence homology with the haemorrhagic toxins from a number of other snake venoms, and some metalloproteinases in the region of the putative Zn-binding sites.
    FEBS Letters 05/1991; 282(1):178-82. · 3.58 Impact Factor

Publication Stats

423 Citations
59.75 Total Impact Points

Institutions

  • 1990–2012
    • Fundação Ezequiel Dias
      Camelleira, Pernambuco, Brazil
  • 2001
    • Universidade Federal do Espírito Santo
      • Departamento de Ciências Fisiológicas
      Vitória, Estado do Espirito Santo, Brazil
  • 1990–2001
    • Durham University
      • School of Biological and Biomedical Sciences
      Durham, England, United Kingdom
  • 1995
    • Federal University of Minas Gerais
      • Departamento de Bioquímica e Imunologia
      Belo Horizonte, Estado de Minas Gerais, Brazil
  • 1992
    • University of Bayreuth
      • Chair of Plant Systematics
      Bayreuth, Bavaria, Germany